Adenovirus 36 (AdV36) causes increased adiposity (leading to obesity) and metabolic changes in animals. Causation in humans has not been established, but the infection is associated with the same changes as seen in animals, i.e. increased adiposity, weight gain, and decreases in serum lipid and fasting glucose. Elucidation of AdV36's role in the current obesity epidemic will rely, in large part, on longitudinal studies o large populations. Also, understanding AdV36's effects may have implications for the planning and success of novel intervention strategies for obesity. Given the strong association of AdV36 and subsequent effects in infected humans, failure to understand AdV36's role in obesity would ignore a key risk factor in obesity and continue to follow an obesity paradigm that has largely failed to lead to effective prevention and control strategies. A major methodological barrier exists to studies of large populations needed to move the field forward. That is, the gold standard and currently the only specific method for detecting AdV36 antibodies is the serum neutralization assay (SNA)-a complicated and time-consuming method that has produced inconsistent results among laboratories. In its place, a rapid, high throughput, easy to perform, and objective assay is urgently needed to open the field to new investigators, advance the pace of current studies, and improve consistency of results. This application will develop an improved assay by identifying specific epitopes on the AdV36 hexon protein and using recombinant proteins and/or peptides in a simple ELISA-based system to detect evidence of AdV36 infection, i.e. specific (neutralizing) antibodies. The AdV hexon protein is involved in initial interactions between the virus and host cell and contains specific neutralization epitopes. The AdV36 hexon sequence has been compared to other adenoviruses, revealing nine hypervariable regions (HVRs) likely to contain one or more specific epitopes. We have cloned and expressed four recombinant fusion proteins containing AdV36 HVRs and tested each using SNA-identified positive or negative sera. Two recombinants show promise for development of a sensitive and specific assay but require further characterization and validation. Further, overlapping peptides based on HVR sequences from these recombinants will be synthesized and tested for use in an assay system. Recombinants and/or peptides identified as containing AdV36-specific epitopes will be used in an ELISA to test 300 individual sera with known SNA titers. Comparison of results between the two test systems will demonstrate the sensitivity and specificity of the new assay. In summary, a sensitive, specific, and rapid assay system that no longer depends on cell culture and live virus is essential. Without an improved methodology, studies of AdV36's role in obesity will be slow, carried out by only a few investigators, and fraught with inter-laboratory variability in results.